Bottom Line:
These data suggest that expression of the JAK2 Deltaexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs.This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis.This mutation is missed if DNA rather than RNA is used for testing.

Background: The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Deltaexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing.

Methodology/principal findings: We investigated the possibility that MPN patients may express the JAK2 Deltaexon14 at low levels (<15% of total transcript) not routinely detectable by RT-PCR with direct sequencing. Using a sensitive RT-PCR-based fluorescent fragment analysis method to quantify JAK2 Deltaexon14 mRNA expression relative to wild-type, we tested 61 patients with confirmed MPNs, 183 with suspected MPNs (93 V617F-positive, 90 V617F-negative), and 46 healthy control subjects. The Deltaexon14 variant was detected in 9 of the 61 (15%) confirmed MPN patients, accounting for 3.96% to 33.85% (mean = 12.04%) of total JAK2 transcript. This variant was also detected in 51 of the 183 patients with suspected MPNs (27%), including 20 of the 93 (22%) with V617F (mean [range] expression = 5.41% [2.13%-26.22%]) and 31 of the 90 (34%) without V617F (mean [range] expression = 3.88% [2.08%-12.22%]). Immunoprecipitation studies demonstrated that patients expressing Deltaexon14 mRNA expressed a corresponding truncated JAK2 protein. The Deltaexon14 variant was not detected in the 46 control subjects.

Conclusions/significance: These data suggest that expression of the JAK2 Deltaexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs. This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis. This mutation is missed if DNA rather than RNA is used for testing.

pone-0012165-g004: Source of RNA for detection of JAK2 Δexon14 transcript using RT-PCR with fragment length analysis.Similar results are obtained whether RNA is isolated from plasma or peripheral blood cells. Size marker is shown as red peaks and amplification products are shown in blue.

Mentions:
When JAK2 RNA (not DNA) is used for direct sequencing, the Δexon 14 transcript is reliably detected if present at levels >15% to 20% of total JAK2 RNA (Figure 1 and 2). MPN patients rarely have Δexon 14 transcript levels above this threshold, and the results of direct sequence analysis can be difficult to interpret in patients with apparent low-level expression. In these cases, the Δexon14 transcript can easily be interpreted as background or poor sequencing if the background sequence is not read completely and aligned to the JAK2 sequence (Figure 2). To more accurately detect low levels of Δexon 14 transcript expression, we developed an RT-PCR–based assay with fluorescent fragment length analysis. With this method, the JAK2 Δexon14 splice variant shows a 185-bp fragment while the wild-type shows a 273-bp fragment (Figure 3). To confirm that the Δexon14 splice variant that is detected in plasma is actually present in cells, we analyzed paired cell and plasma RNA from patients previously confirmed to show expression of the Δexon14 transcript in plasma. Both plasma and cells revealed reliable results for detecting Δexon14 transcripts (Figure 4).

pone-0012165-g004: Source of RNA for detection of JAK2 Δexon14 transcript using RT-PCR with fragment length analysis.Similar results are obtained whether RNA is isolated from plasma or peripheral blood cells. Size marker is shown as red peaks and amplification products are shown in blue.

Mentions:
When JAK2 RNA (not DNA) is used for direct sequencing, the Δexon 14 transcript is reliably detected if present at levels >15% to 20% of total JAK2 RNA (Figure 1 and 2). MPN patients rarely have Δexon 14 transcript levels above this threshold, and the results of direct sequence analysis can be difficult to interpret in patients with apparent low-level expression. In these cases, the Δexon14 transcript can easily be interpreted as background or poor sequencing if the background sequence is not read completely and aligned to the JAK2 sequence (Figure 2). To more accurately detect low levels of Δexon 14 transcript expression, we developed an RT-PCR–based assay with fluorescent fragment length analysis. With this method, the JAK2 Δexon14 splice variant shows a 185-bp fragment while the wild-type shows a 273-bp fragment (Figure 3). To confirm that the Δexon14 splice variant that is detected in plasma is actually present in cells, we analyzed paired cell and plasma RNA from patients previously confirmed to show expression of the Δexon14 transcript in plasma. Both plasma and cells revealed reliable results for detecting Δexon14 transcripts (Figure 4).

Bottom Line:
These data suggest that expression of the JAK2 Deltaexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs.This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis.This mutation is missed if DNA rather than RNA is used for testing.

Background: The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Deltaexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing.

Methodology/principal findings: We investigated the possibility that MPN patients may express the JAK2 Deltaexon14 at low levels (<15% of total transcript) not routinely detectable by RT-PCR with direct sequencing. Using a sensitive RT-PCR-based fluorescent fragment analysis method to quantify JAK2 Deltaexon14 mRNA expression relative to wild-type, we tested 61 patients with confirmed MPNs, 183 with suspected MPNs (93 V617F-positive, 90 V617F-negative), and 46 healthy control subjects. The Deltaexon14 variant was detected in 9 of the 61 (15%) confirmed MPN patients, accounting for 3.96% to 33.85% (mean = 12.04%) of total JAK2 transcript. This variant was also detected in 51 of the 183 patients with suspected MPNs (27%), including 20 of the 93 (22%) with V617F (mean [range] expression = 5.41% [2.13%-26.22%]) and 31 of the 90 (34%) without V617F (mean [range] expression = 3.88% [2.08%-12.22%]). Immunoprecipitation studies demonstrated that patients expressing Deltaexon14 mRNA expressed a corresponding truncated JAK2 protein. The Deltaexon14 variant was not detected in the 46 control subjects.

Conclusions/significance: These data suggest that expression of the JAK2 Deltaexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs. This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis. This mutation is missed if DNA rather than RNA is used for testing.